Increased hydrogen peroxide (HOOH) is thought to occur under conditions of tissue reperfusion with oxygenated media after hypoxia. We were first to show that when modified hemoglobins are challenged with HOOH, damaging and potentially toxic species are formed including the ferryl protein radical (Fe+4). Since the vascular endothelium is the first in line to exposure of circulating hemoglobin-based blood substitutes, we investigated the effects of a variety of modified hemoglobins on the integrity of bovine aortic endothelial cells (BAEC) and on the HOOH mediated cytotoxicity. We found unmodified hemoglobin AO to be protective against peroxide-induced endothelial cell necrosis, probably due to its peroxidase activity (cycling between the ferric and the ferryl heme in the presence of HOOCH can result in apparent peroxidase activity), and that the level of activity was also dependant on structural modification of the protein. Intra- and intermolecular cross-linked hemoglobin, however, did not protect against peroxide-induced endothelial cell damage or production of the ferryl species. We have also developed an endothelial cell model of ischemia and reperfusion (I/R) with regard to cell death, H2O2 production, nitrate/nitrite production, and lipid peroxidation. Hemoglobin was found to decrease the concentration of H2O2 in cell media, while it increased cellular lipid peroxidation and had no effect on the number of cells surviving ischemia after 24 hours. Endothelial cells were also found to have an effect on the redox state of hemoglobin. Site directed mutagenesis is a potentially effective tool for engineering hemoglobin because it allows the fine tuning of protein function and stability. At this point, myoglobin has provided a simple prototype for these experiments, and indeed a number of myoglobin mutants have been prepared in collaboration with Rice University that have different ligand binding, autoxidation and stability. We have data to show clear differences among these myoglobins in terms of the rates of ferryl Hb formation and its persistence in solutions and that selectively constructed mutants will be used for probing the mechanism of HOOH mode of entry into the proteins. This will ultimately help in the design of a protein that can stereochemically restrict the entry of HOOCH and minimize its oxidative effect.
